Isolation of<i>Bluetongue virus</i>from canine abortions

Dubovi, Edward J.; Hawkins, Melissa; Griffin, Robert; Johnson, Donna J.; Ostlund, Eileen N.

doi:10.1177/1040638713489982

Cited by 16 publications

(25 citation statements)

References 8 publications

(12 reference statements)

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“…Ten percent tissue pools of lung, liver, spleen, and intestine from puppy 2, intestine from puppy 4a, lung and intestine from puppy 4b, and lung, liver, spleen, kidney, and brain from puppy 8 were prepared in Eagle's minimal essential medium (MEM-E) containing 0.5% bovine serum albumin and 10 g/ml ciprofloxacin. After tissue disruption and low-speed centrifugation, 1 ml of the filtered supernatants from puppies 2, 4a, and 4b were inoculated onto monolayers of canine fibroblast-like A-72 cells (ATCC CRL-1542) and immortalized canine kidney cells (AHDC, Cornell University), while supernatant from puppy 8 was inoculated onto immortalized canine kidney cells and human colorectal adenocarcinoma HRT-18 (ATCC CCL-244) cells grown in 25-cm 2 flasks as previously described (34). Supernatants from puppies 4a and 4b also were inoculated onto canine kidney MDCK cells (ATCC CCL-34) and HRT-18 cells, respectively.…”

Section: Methodsmentioning

confidence: 99%

Genotypic Characterization of Canine Coronaviruses Associated with Fatal Canine Neonatal Enteritis in the United States

et al. 2014

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Emerging canine coronavirus (CCoV) variants that are associated with systemic infections have been reported in the European Union; however, CCoV-associated disease in the United States is incompletely characterized. The purpose of this study was to correlate the clinicopathological findings and viral antigen distribution with the genotypic characteristics of CCoV in 11 puppies from nine premises in five states that were submitted for diagnostic investigation at Cornell University between 2008 and 2013. CCoV antigen was found in epithelial cells of small intestinal villi in all puppies and the colon in 2 of the 10 puppies where colon specimens were available. No evidence of systemic CCoV infection was found. Comparative sequence analyses of viral RNA extracted from intestinal tissues revealed CCoV-II genotype in 9 out of 11 puppies. Of the nine CCoV-IIs, five were subtyped as group IIa and one as IIb, while three CCoVs could not be subtyped. One of the CCoV-IIa variants was isolated in cell culture. Infection with CCoV alone was found in five puppies, of which two also had small intestinal intussusception. Concurrent infections with either parvovirus (n ‫؍‬ 1), attaching-effacing Escherichia coli (n ‫؍‬ 4), or protozoan parasites (n ‫؍‬ 3) were found in the other six puppies. CCoV is an important differential diagnosis in outbreaks of severe enterocolitis among puppies between 4 days and 21 weeks of age that are housed at high population density. These findings will assist with the rapid laboratory diagnosis of enteritis in puppies and highlight the need for continued surveillance for CCoV variants and intestinal viral diseases of global significance.

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Section: Methodsmentioning

confidence: 99%

Genotypic Characterization of Canine Coronaviruses Associated with Fatal Canine Neonatal Enteritis in the United States

et al. 2014

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“…Bluetongue virus (BTV) is a double‐stranded RNA virus that belongs to the genus Orbivirus , family Reoviridae (Attoui, Maan, Anthony, & Mertens, ). The virus is predominantly transmitted between mammalian hosts by Culicoides biting midges (Koenraadt et al, ), but in some cases it can be transmitted vertically (Chauhan et al, ; Dubovi, Hawkins, Griffin, Johnson, & Ostlund, ) or horizontally between animals through ingestion of infected tissues, such as during fights over hierarchy (López‐Olvera et al, ), by eating infected placenta (Menzies et al, ), ingestion of virus‐contaminated colostrum by newborn ruminants (Backx, Heutink, Rooij, & Rijn, ), and mechanically through reused needles (Darpel et al, ). Bluetongue disease has been reported from Australia, North and South America, Africa, the Middle East, Asia and Europe (Maclachlan & Mayo, ).…”

Section: Introductionmentioning

confidence: 99%

Characterization of bluetongue virus serotype 28

Bumbarov¹,

Golender²,

Jenckel

et al. 2019

Transbound Emerg Dis

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Bluetongue virus (Reoviridae; Orbivirus, BTV), which is usually transmitted by biting midges, affects wild and domestic ruminants worldwide, thereby causing an economically important disease. Recently, a putative new BTV strain was isolated from contaminated vaccine batches. In this study, we investigated the genomic and clinical characteristics of this isolate, provisionally designated BTV‐28. Phylogenetic analysis of BTV‐28 segment 2 (Seg‐2) showed that it is related to Seg‐2 from BTV serotypes 4, 10, 11, 17, 20 and 24, sharing 64%–66% identity in nucleotide sequences (nt) and 59%–62% in amino acid (aa) sequences of BTV VP2. BTV‐28 Seg‐6 is related to the newly reported XJ1407 BTV isolate, sharing 76.70% nt and 90.87% aa sequence identity. Seg‐5 was most closely related to a South African BTV‐4 strain, and all other segments showed close similarity to BTV‐26. Experimental infection by injection of 6‐month‐old ewes caused clinical signs in all injected animals, lasting from 2 to 3 days to several weeks post‐infection, including high body temperature, conjunctivitis, nasal discharge and rhinitis, facial oedema, oral hyperaemia, coronitis, cough, depression and tongue cyanosis. Naïve control animals, placed together with the infected sheep, displayed clinical signs and were positive for viral RNA, but their acute disease phase was shorter than that of BTV‐injected ewes. Control animals that were kept in a separated pen did not display any clinical signs and were negative for viral RNA presence throughout the experiment. Seroconversion was observed in the injected and in one of the two contact‐infected animals. These findings demonstrate that BTV‐28 infection of sheep can result in clinical manifestation, and the clinical signs detected in the contact animals suggest that it might be directly transmitted between the mammalian hosts.

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“…The manifestation of the disease depends on serotype, species, breed, and age of animal with morbidity that could reach up to 100% and mortality between 30% and 70% [ 5 ]. Through transplacental transfer, the virus is also capable of exerting teratogenic effects, or the infection may lead to abortion in the pregnant host [ 6 , 7 ]. Curtailing the initial introduction into regions which harbor susceptible host and vector species and vaccination of the susceptible animal species may aid in effective prevention and control of BT [ 8 ].…”

Section: Introductionmentioning

confidence: 99%

Standardization and application of real-time polymerase chain reaction for rapid detection of bluetongue virus

et al. 2018

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AimThe present study was designed to standardize real-time polymerase chain reaction (PCR) for detecting the bluetongue virus from blood samples of sheep collected during outbreaks of bluetongue disease in the year 2014 in Andhra Pradesh and Telangana states of India.Materials and MethodsA 10-fold serial dilution of Plasmid PUC59 with bluetongue virus (BTV) NS3 insert was used to plot the standard curve. BHK-21 and KC cells were used for in vitro propagation of virus BTV-9 at a TCID50/ml of 105 ml and RNA was isolated by the Trizol method. Both reverse transcription-PCR and real-time PCR using TaqMan probe were carried out with RNA extracted from virus-spiked culture medium and blood to compare the sensitivity by means of finding out the limit of detection (LoD). The results were verified by inoculating the detected and undetected dilutions onto cell cultures with further cytological (cytopathic effect) and molecular confirmation (by BTV-NS1 group-specific PCR). The standardized technique was then applied to field samples (blood) for detecting BTV.ResultsThe slope of the standard curve obtained was −3.23, and the efficiency was 103%. The LoD with RT-PCR was 8.269E×103 number of copies of plasmid, whereas it was 13 with real-time PCR for plasmid dilutions. Similarly, LoD was determined for virus-spiked culture medium, and blood with both the types of PCR and the values were 103 TCID 50/ml and 104 TCID 50/ml with RT-PCR and 10° TCID 50/ml and 102 TCID 50/ml with real-time PCR, respectively. The standardized technique was applied to blood samples collected from BTV suspected animals; 10 among 20 samples were found positive with Cq values ranging from 27 to 39. The Cq value exhibiting samples were further processed in cell cultures and were confirmed to be BT positive. Likewise, Cq undetected samples on processing in cell cultures turned out to be BTV negative.ConclusionReal-time PCR was found to be a very sensitive as well as reliable method to detect BTV present in different types of samples, including blood samples collected from BTV-infected sheep, compared to RT-PCR. The LoD of BTV is likely influenced by sample type, possibly by the interference by the other components present in the sample.

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Isolation ofBluetongue virusfrom canine abortions

Cited by 16 publications

References 8 publications

Genotypic Characterization of Canine Coronaviruses Associated with Fatal Canine Neonatal Enteritis in the United States

Genotypic Characterization of Canine Coronaviruses Associated with Fatal Canine Neonatal Enteritis in the United States

Characterization of bluetongue virus serotype 28

Standardization and application of real-time polymerase chain reaction for rapid detection of bluetongue virus

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